Project Summary/Abstract The process of craniofacial morphogenesis demands the coordinated outgrowth of multiple facial prominences that are initially spatially separated. Underscoring this complex choreography, 1/3 of all human birth defects affect craniofacial development. Specifically, cleft lip with or without cleft palate (CL/P), the most common craniofacial birth abnormality that affects the midface, occurs in 1/500-700 live births. During embryonic development, regulatory genes such as Satb2 and Pbx1 control the dynamics of transcriptional activity, tissue- level patterning, and intercellular signaling to modulate the timing of progenitor proliferation, cell cycle exit, and osteoblast differentiation. Variation in these dynamics underpins both evolutionary variation in midfacial outgrowth and the etiology of craniofacial birth defects. Notably, it has been established that the main variable driving species-specific midfacial length and morphology (prognathism) is the maxillary bone of the upper jaw. Despite the availability of large datasets of regulatory elements generated from mouse, chimp, and human facial tissues and cranial neural crest cells, and despite studies of craniofacial phenotypes in mutant mice, the majority of regulatory networks that control embryonic midfacial outgrowth are still unknown with respect to their spatiotemporal context-dependence during midfacial morphogenesis. This knowledge gap negatively impacts the efforts of human geneticists to identify and map causal mutations associated with human craniofacial pathologies. The goal of these studies is to define the cis-regulatory landscapes underpinning midfacial outgrowth in order to understand how morphogenesis of the craniofacial complex as a whole is coordinated with variation in the growth of specific skeletal elements. This project will employ a vertical gene- centric approach to test the hypothesis that a Satb2/Pbx-dependent regulatory network coordinates morphogenetic cell behaviors and osteoblast differentiation critical for normal maxillary outgrowth. Using available Satb2 and Pbx mutant mice, Aim1a will define the regulatory hierarchy of these genes and their impact on maxillary morphogenesis; Aim1b will establish Satb2/Pbx-dependent genomic networks that drive maxillary outgrowth and differentiation by RNA-Seq assays. Moreover, the hypothesis will be tested that regulatory landscapes of genes critical for craniofacial development show distinct spatiotemporal activity in C57Bl/6J (B6) and PWK/PhJ (PWK) mice, two genetically distinct mouse strains with varying maxillary length. In a parallel genome-wide approach that leverages sequence variation in these two mouse lines Aim2a will produce microCT based morphometry on B6 vs PWK embryonic heads to define their respective maxillary growth trajectories; Aim2b will generate genome-wide annotation of the transcriptional regulatory landscapes in developing skeletal anlagen of mice with different degrees of prognathism by GRO-seq analyses on B6 vs PWK embryonic maxillae. These studies will ultimately define the mechanisms underlying tolerance to morphological variation that separate normal craniofacial development from pathology.